Active Projects
Structures of Protein Complexes Regulating Transcription in Embryonic Stem Cells
The major goal of this proposal is to reveal molecular mechanisms underlying formation and function of critical transcriptional assemblies essential to embryonic stem (ES) cells and cells with induced pluripotency (induced pluripotent stem (iPS) cells). The proposed structural and functional studies will propel our general knowledge of the basic mechanisms controlling cell fate, including those underlying self renewal, differentiation and pathogenesis of cancer, and would have a major impact on stem cell research as well as regenerative medicine.
This work is in concert with Paul Webb, The Methodist Hospital Research Institute, Houston, Bruce Conklin and Shinya Yamanaka, of the Gladstone Institute of Cardiovascular Disease, San Francisco and Ian Wilson, The Joint Center for Stuctural Genomics, La Jolla, CA.
NIH U01 GM094614, Consortia for High-Throughput-Enabled Structural Biology Partnerships. This program is part of NIH's Protein Structure Initative: Biology Program
Selective thyroid hormone analogs for metabolic syndrome
We plan to investigate biochemical and structural aspects of key NR/cofactor interactions that are obligate steps for maturation of the receptor complexes in the cytoplasm and their translocation into the nucleus and whether it will become possible to control receptor activity with small molecules that bind directly to receptor surfaces involved in these events.
NIH RC4 DK090849, subcontract with Paul Webb, TMHRI, Houston
Screening for antagonists of nuclear receptor LRH-1 in pancreatic cancer cells
We propose to discover selective inhibitors of LRH-1 activity that would arrest pancreatic cancer cell proliferation.
NIH R03 MH094165
Mechanisms of Regulation of LRH-1, Nanog and SF-1
by DAX-1
The
goal of this project is to understand the principles and atomic level
details of the mechanisms of regulation of LRH-1 and SF-1 by DAX-1. Combined biochemical and structural analyses that we propose in this grant application will provide necessary information for understanding how LRH-1, SF-1, Dax-1 and Nanog function, how their activities are regulated, and how one could design and use specific modulators to tune aberrant activities hich underlie endocrine metabolic diseases and cancer. In addition, this work will show how we might elucidate the role of Dax-1 in regulating other transcription factors including TIF1, Rif1, Pelo, REST, Sall4, Sp1, Zfp609 and other nuclear receptors including Nur77, ERR2, ER, AR, HNF4 and GCNF.
NIH R01 DK078075 and NIH Diversity Supplement to Leslie Cruz, Graduate Student
Nuclear Receptor LRH-1 as a Target in Pancreatic Cancer
The main goal of this project is to explore function of an essential regulator of pancreatic development, the nuclear receptor LRH-1, in pancreatic cancer cells, and discover compounds that inhibit the LRH-1 activity in pancreatic cancer cells.NIH R21 CA140751
Selective Thyroid Hormone Analogs for Metabolic Syndrome
We plan to investigate biochemical and structural aspects of key NR/cofactor interactions that are obligate steps for maturation of the receptor complexes in the cytoplasm and their translocation into the nucleus and whether it will become possible to control receptor activity with small molecules that bind directly to receptor surfaces involved in these events.
RC4 DK090849, sub-project with Paul Webb
Imaging Nuclear Receptor LRH-1 in Functional Transcriptional Assemblies
Cellular transcription programs require that many proteins of different function assemble on chromatin in response to signals that originate outside the nucleus. The largest family of proteins initiating these transcriptional processes is that of the nuclear receptors. The nuclear receptors are multidomain proteins, and there are no atomic resolution images of their complete structures. Our proposal focuses on the nuclear receptor LRH-1 (Liver Receptor Homologue 1, termed also Pancreas Receptor Homologue 1), a critical transcription factor found in liver, intestines and pancreas. We chose LRH-1 as a primary target because of the receptor’s critical roles in human developmental, metabolic and numerous pathophysiological processes.Our ultimate goal is to image the full length LRH-1 receptor with partner co-regulatory proteins when bound to DNA. We will use an advanced methodology to prepare and assemble the multiple domains of LRH-1 with partner proteins and specific DNA fragments so that the transcriptional assembly would be ordered well enough for analysis by X-ray crystallography. We plan to employ a stabilizing transcriptional partner protein, beta-catenin, that will permit biochemical preparation of previously intractable molecules and mediate their interactions in the functional complexes. We will use a systematic approach to identify stable assemblies of LRH-1 with its characterized transcriptional regulators. Specific DNA fragments representing known response elements of LRH-1 will be included in evaluation of our assemblies. Methods that we develop for imaging the regulatory complexes built by LRH-1 will be directly applicable to other nuclear receptors and their transcriptional assemblies. Our goal is to take a major step in learning how to determine structures for functional nuclear receptors andlearn atomic level details about the mechanisms of their assembly and regulation of transcription.
NIH / NIDDK (ARRA award) R21 DK084504
Novel Inhibitors of Nuclear Receptor Function
Objectives are development of novel "drug-like" inhibitors that specifically prevent the interaction of nuclear receptors with their coregulating proteins through their NR box binding site, and functionally block transcriptional activation by nuclear receptors. The research design integrates knowledge based structure, chemistry, and cell biology to rationally produce novel small molecule inhibitors of the targeted protein interaction. Methods are high throughput screening, parallel chemistry, medicinal chemistry, high throughput X-ray crystallography (structural genomics), cell biology, genomics, and pharmacology.
NIH RO1 (R.K. Guy, St. Judes)
Thyroid Hormone Receptor - X-Ray Crystallography
Aims are to determine the X-ray crystal structure of the hTRß LBD bound to several different ligands that perturb the structure in different ways, and to determine X-ray structures of RXR and TR DBD-LBD proteins and the full-length TR. The new structures should provide information about hormone-induced conformational changes, mechanisms of receptor interactions with other proteins, the multiple receptor domains, and relations between domains. The results should yield insights into TR function and how ligands act as agonists or antagonists.
NIH R01 DK041842 (J. Baxter, TMHRI)
Structure and Function of Molecular Motors
Project V: Structural Studies on Motor Proteins. This project moves the focus of the X-ray crystallography component of this program project from analysis of the motility mechanisms of molecular motors towards an atomic level analysis of their regulation and attachments to protein partners. We plan to define the role of the neck domain in the kinesin homolog KinI which drives the remarkable catastrophic breakdown of tubulin, to learn how Ca ion regulates kinesin binding to microtubules, and to reveal how the tubulin dimer affects KinI function. We will crystallize the catalytic domain of myosin with a fragment of an actin polymer, to compare myosin-track interactions with kinesin-track interactions. Finally we will characterize the binding domain that connects vesicles to the myosin motor of myosin V, a vesicle transporter in neurons. NIH Program Project (Roger Cooke, PI)
